Determination of tissue contributions to the circulating lipid pool in cold exposure via systematic assessment of lipid profiles

Plasma lipid levels are altered in chronic conditions such as type 2 diabetes and cardiovascular disease as well as during acute stresses such as fasting and cold exposure. Advances in MS-based lipidomics have uncovered a complex plasma lipidome of more than 500 lipids that serve functional roles, including as energy substrates and signaling molecules. This plasma lipid pool is maintained through regulation of tissue production, secretion, and uptake. A major challenge in understanding the lipidome complexity is establishing the tissues of origin and uptake for various plasma lipids, which is valuable for determining lipid functions. Using cold exposure as an acute stress, we performed global lipidomics on plasma and in nine tissues that may contribute to the circulating lipid pool. We found that numerous species of plasma acylcarnitines (ACars) and ceramides (Cers) were significantly altered upon cold exposure. Through computational assessment, we identified the liver and brown adipose tissue as major contributors and consumers of circulating ACars, in agreement with our previous work. We further identified the kidney and intestine as novel contributors to the circulating ACar pool and validated these findings with gene expression analysis. Regression analysis also identified that the brown adipose tissue and kidney are interactors with the plasma Cer pool. Taken together, these studies provide an adaptable computational tool to assess tissue contribution to the plasma lipid pool. Our findings have further implications in understanding the function of plasma ACars and Cers, which are elevated in metabolic diseases.     

A comprehensive classification system for lipids

Lipids are produced, transported, and recognized by the concerted actions of numerous enzymes, binding proteins, and receptors. A comprehensive analysis of lipid molecules, "lipidomics," in the context of genomics and proteomics is crucial to understanding cellular physiology and pathology; consequently, lipid biology has become a major research target of the postgenomic revolution and systems biology. To facilitate international communication about lipids, a comprehensive classification of lipids with a common platform that is compatible with informatics requirements has been developed to deal with the massive amounts of data that will be generated by our lipid community. As an initial step in this development, we divide lipids into eight categories (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides) containing distinct classes and subclasses of molecules, devise a common manner of representing the chemical structures of individual lipids and their derivatives, and provide a 12 digit identifier for each unique lipid molecule. The lipid classification scheme is chemically based and driven by the distinct hydrophobic and hydrophilic elements that compose the lipid. This structured vocabulary will facilitate the systematization of lipid biology and enable the cataloging of lipids and their properties in a way that is compatible with other macromolecular databases.     

Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels

Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes. Currently, high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC ESI-MS/MS) offers the most efficient method of quantifying C1P. However, the published protocols have several drawbacks causing overestimations and carryovers. Here, the reported overestimation of C1P was shown to be due to incomplete neutralization of base hydrolyzed lipid extracts leading to the hydrolysis of SM to C1P. Actual quantity of C1P in cells (6 pmols/10⁶ cells) was much lower than previously reported. Also, the major species of C1P produced by ceramide kinase (CERK) was found to be d_18:1/16:0 with a minority of d_18:1/24:1 and d_18:1/24:0. The artifactual production of C1P from SM was used for generating C1Ps as retention time markers. Elimination of carryovers between samples and a 2-fold enhancement in the signal strength was achieved by heating the chromatographic column to 60^°C. The role of ceramide transport protein (CERT) in supplying substrate to CERK was also revalidated using this new assay. Finally, our results demonstrate the presence of additional pathway(s) for generation of the C1P subspecies, d_18:1/18:0 C1P, as well as a significant portion of d_18:1/16:0, d_18:1/24:1, and d_18:1/24:0. In conclusion, this study introduces a much improved and validated method for detection of C1P by mass spectrometry and demonstrates specific changes in the C1P subspecies profiles upon downregulation of CERK and CERT.     

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method

Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 μl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1–21.4 μM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.     

Isolation of lipids from biological samples

Abstract

Isolation of the lipid fraction from biological samples has been a crucial part of countless studies over the last century. This considerable research interest has led to the development of a number of methods for isolating a range of molecular species that fall under the umbrella term “lipid”. Such methods vary in popularity, complexity, specificity and even toxicity. In this review, we explore examples of published methods (1952–2014) for isolating lipids from biological samples and attempt to assess the limits of techniques both from a chemical and biological perspective. We also suggest how a suitable method might be chosen for a novel application.     

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway

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Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes

Shotgun lipidomics, comprised of intrasource separation, multidimensional mass spectrometry and computer-assisted array analysis, is an emerging powerful technique in lipidomics. Through effective intrasource separation of predetermined groups of lipid classes based on their intrinsic electrical propensities, analyses of lipids from crude extracts of biologic samples can be directly and routinely performed. Appropriate multidimensional array analysis of lipid pseudomolecular ions and fragments can be performed leading to the identification and quantitation of targeted lipid molecular species. Since most biologic lipids are linear combinations of aliphatic chains, backbones and head groups, a rich repertoire of multiple lipid building blocks present in discrete combinations represent experimental observables that can be computer reconstructed in conjunction with their pseudomolecular ions to directly determine the lipid molecular structures from a lipid extract. Through this approach, dramatic increases in the accessible dynamic range for ratiometric quantitation and discrimination of isobaric molecular species can be achieved without any prior column chromatography or operator-dependent supervision. At its current state of development, shotgun lipidomics can analyze over 20 lipid classes, hundreds of lipid molecular species and more than 95% of the mass content of a cellular lipidome. Thus, understanding the biochemical mechanisms underlying lipid-mediated disease states will be greatly facilitated by the power of shotgun lipidomics.